This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Fatty acid synthase (FASN) is the enzyme that catalyzes the de novo synthesis of fatty acids in cells. FASN is up-regulated in solid tumors, and its overexpression has been associated with poor prognosis in breast cancer patients. FASN regulates the expression of the human epidermal growth factor receptor 2 (her-2 also known as c-erbB-2) gene. We have recently found that FASN forms a complex with phosphorylated EGFR and HER-2. Our hypothesis is that FASN phosphorylation is critical for HER-2 signaling in breast cancer cells. The specific aim of this proposal is to identify the FASN/HER-2 and FASN/EGFR phosphorylation sites in HER2-overexpressing breast cancer cells using immunoprecipitation, immobilized metal-ion affinity chromatography (IMAC), and high resolution LC-MS/MS.